The effects of oxidative stress on parkin and other E3 ligases

Autosomal recessive mutations within the Parkin gene are associated with degeneration of the substantia nigra and locus coeruleus and an inherited form of Parkinson's disease (PD). As loss-of-function mutations in parkin are responsible for a familial variant of PD, conditions that affect wild-type parkin are likely to be associated with increased risk of idiopathic disease. Previous studies uncovered a unique vulnerability of the parkin protein to dopamine (DA)-induced aggregation and inactivation. In this study, we compared several proteins that share structural elements or ubiquitinating activity with parkin. We report that oxidative stress in several cell lines and primary neurons induces the aggregation of parkin into high molecular weight species, at least a portion of which are self-associated homo-multimers. While parkin was preferentially affected by excess DA, each of the E3 proteins tested were made more insoluble by oxidative stress, and they varied in degree of susceptibility (e.g. parkin > HHARI ≅ CHIP > c-Cbl > E6AP). These conditions of oxidative stress were also associated with decreased parkin E3 ligase activity. Similar to recently conducted studies on α-synuclein processing, both macroautophagy and the proteasome participate in parkin degradation, with the proteasome playing the predominant role for normal parkin turnover and macroautophagy being more important in the degradation of aggregated parkin. These data further highlight the selective vulnerability of parkin to DA-induced modifications, demonstrating for the first time the ability of both endogenous and ectopically expressed parkin to transition into an insoluble state in part through self-association and oligomer formation.     

Cloning of rat parkin cDNA and distribution of parkin in rat brain

The rat parkin cDNA sequence was characterized after screening a rat hypothalamus cDNA library with a 32P-labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465-amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N-terminal ubiquitin and the ring-IBR (in between ring)-ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity-purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295-311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52-kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin-immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin-enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early-onset parkinsonism.     

Astrocytic but not neuronal increased expression and redistribution of parkin during unfolded protein stress

Parkin is a ubiquitin ligase that facilitates proteasomal protein degradation and is involved in a common autosomal recessive form of Parkinson's disease. Its expression is part of the unfolded protein response in cell lines where its overexpression protects against unfolded protein stress. How parkin expression is regulated in brain primary cells under stress situations is however, less well established. Here, the cellular and subcellular localization of parkin under basal conditions and during unfolded protein stress was investigated in primary cultures of rat astrocytes and hippocampal neurons. Immunofluorescense microscopy and biochemical analysis demonstrated that parkin is mainly associated with the endoplasmic reticulum (ER) in hippocampal neurons while it is associated with Golgi membranes, the nuclei and light vesicles in astrocytes. The constitutive parkin expression was high in neurons as compared with astrocytes. However, unfolded protein stress elicited a selective increase in astrocytic parkin expression and a change in distribution, whereas neuronal parkin remained largely unmodified. The cell specific differences argue in favour of different cellular binding sites and substrates for the protein and a pathogenic role for astrocytes in Parkinson's disease caused by parkin dysfunction.     

Alterations in the solubility and intracellular localization of parkin by several familial Parkinson's disease-linked point mutations

Mutations in the parkin gene, which encodes a ubiquitin ligase, are currently recognized as the main contributor to familial forms of Parkinson's disease (PD). A simple assumption about the effects of PD-linked mutations in parkin is that they impair or ablate the enzyme activity. However, a number of recent studies, including ours, have indicated that many disease-linked point mutants of parkin retain substantial catalytic activity. To understand how the plethora of mutations on parkin contribute to its dysfunction, we have conducted a systematic analysis of a significant number of parkin point mutants (22 in total), which represent the majority of parkin missense/nonsense mutations reported to date. We found that more than half of these mutations, including many located outside of the parkin RING fingers, produce alteration in the solubility of parkin which influences its detergent extraction property. This mutation-mediated alteration in parkin solubility is also associated with its propensity to form intracellular, aggresome-like, protein aggregates. However, they do not represent sites where parkin substrates become sequestered. As protein aggregation sequesters the functional forms away from their normal sites of action, our results suggest that alterations in parkin solubility and intracellular localization may underlie the molecular basis of the loss of function caused by several of its mutations.     

Methamphetamine oxidatively damages parkin and decreases the activity of 26S proteasome in vivo

Methamphetamine (METH) is toxic to dopaminergic (DAergic) terminals in animals and humans. An early event in METH neurotoxicity is an oxidative stress followed by damage to proteins and lipids. The removal of damaged proteins is accomplished by the ubiquitin-proteasome system (UPS) and the impairment of this system can cause neurodegeneration. Whether dysfunction of the UPS contributes to METH toxicity to DAergic terminals has not been determined. The present investigation examined the effects of METH on functions of parkin and proteasome in rat striatal synaptosomes. METH rapidly modified parkin via conjugation with 4-hydroxy-2-nonenal (4-HNE) to decrease parkin levels and decreased the activity of the 26S proteasome while simultaneously increasing chymotrypsin-like activity and 20S proteasome levels. Prior injections of vitamin E diminished METH-induced changes to parkin and the 26S proteasome as well as long-term decreases in DA and its metabolites' concentrations in striatal tissue. These results suggest that METH causes lipid peroxidation-mediated damage to parkin and the 26S proteasome. As the changes in parkin and 26S occur before the sustained deficits in DAergic markers, an early loss of UPS function may be important in mediating the long-term degeneration of striatal DAergic terminals via toxic accumulation of parkin substrates and damaged proteins.     

Neuronal NLRP3 is a parkin substrate that drives neurodegeneration in Parkinson’s disease

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Cell death mechanisms in neurodegeneration

Progressive cell loss in specific neuronal populations often associated with typical cytoskeletal protein aggregations is a pathological hallmark of neurodegenerative disorders, but the nature, time course and molecular causes of cell death and their relation to cytoskeletal pathologies are still unresolved. Apoptosis or alternative pathways of cell death have been discussed in Alzheimer's disease and other neurodegenerative disorders. Apoptotic DNA fragmentation in human brain as a sign of neuronal injury is found too frequent as to account for continous neuron loss in these slowly progressive processes. Morphological studies revealed extremely rare apoptotic neuronal death in Alzheimer's disease but yielded mixed results for Parkinson's disease and other neurodegenerative disorders. Based on recent data in human brain, as well as in animal and cell culture models, a picture is beginning to emerge suggesting that, in addition to apoptosis, other forms of programmed cell death may participate in neurodegeneration. Better understanding of the molecular players will further elucidate the mechanisms of cell death in these disorders and their relations to cytoskeletal abnormalities. Susceptible cell populations in a proapoptotic environment show increased vulnerability towards multiple noxious factors discussed in the pathogenesis of neurodegeneration. In conclusion, although many in vivo and in vitro data are in favor of apoptosis involvement in neurodegenerative processes, there is considerable evidence that very complex events may contribute to neuronal death with possible repair mechanisms, the elucidation of which may prove useful for future prevention and therapy of neurodegenerative disorders.     

Parkin Transcript Variants in Rat and Human Brain

Alternative splicing has an important role in expanding protein diversity. We have identified complementary DNA species from adult rat and fetal human brain encoding seven new splice variants of parkin, a gene mutated in autosomal recessive juvenile parkinsonism (ARJP). Alternative splicing affects almost all previously characterized exons, plus 3 new exons of 72, 156, and 180 nucleotides. This creates the potential to express hundreds of different isoforms. The encoded parkin isoforms have different amino acid composition, post-translational modifications, and, most important, molecular architectures. They diverge for the presence or absence of the ubiquitin-like domain, one or two C3HC4 ring fingers, the in-between ring fingers (IBR) domain, and a thiol proteases active site, which has not been previously characterized. Distinct expression patterns occur in primary cultures of neuronal and glial cells. Extensive splicing of parkin produces regional and structural diversity and may have important implications for the pathogenetic mechanisms underlying ARJP.     

USP8 regulates mitophagy by removing K6‐linked ubiquitin conjugates from parkin

Mutations in the Park2 gene, encoding the E3 ubiquitin‐ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin‐mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin‐mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin‐mediated mitophagy. USP8 preferentially removes non‐canonical K6‐linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8‐mediated deubiquitination of K6‐linked ubiquitin conjugates from parkin in mitochondrial quality control.     

Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice

Recessively inherited loss-of-function mutations in the parkin , DJ-1 , or PINK1 gene are linked to familial cases of early-onset Parkinson's diseases (PD), and heterozygous mutations are associated with increased incidence of late-onset PD. We previously reported that single knockout mice lacking Parkin, DJ-1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ-1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila .     

Adaptation to chronic MG132 reduces oxidative toxicity by a CuZnSOD-dependent mechanism

To study whether and how cells adapt to chronic cellular stress, we exposed PC12 cells to the proteasome inhibitor MG132 (0.1 μM) for 2 weeks and longer. This treatment reduced chymotrypsin-like proteasome activity by 47% and was associated with protection against both 6-hydroxydopamine (6-OHDA; 100 μM) and higher dose MG132 (40 μM). Protection developed slowly over the course of the first 2 weeks of exposure and was chronic thereafter. There was no change in total GSH levels after MG132. Buthionine sulfoximine (100 μM) reduced GSH levels by 60%, but exacerbated 6-OHDA toxicity to the same extent in both MG132-treated and control cells and failed to reduce MG132-induced protection. Chronic MG132 resulted in elevated antioxidant proteins CuZn superoxide dismutase (SOD; +55%), MnSOD (+21%), and catalase (+15%), as well as chaperone heat-shock protein 70 (+42%). Examination of SOD enzyme activity revealed higher levels of CuZnSOD (+40%), with no change in MnSOD. We further assessed the mechanism of protection by reducing CuZnSOD levels with two independent siRNA sequences, both of which successfully attenuated protection against 6-OHDA. Previous reports suggested that artificial over-expression of CuZnSOD in dopaminergic cells is protective. Our data complement such observations, revealing that dopaminergic cells are also able to use endogenous CuZnSOD in self-defensive adaptations to chronic stress, and that they can even do so in the face of extensive GSH loss.     

Disease-associated protein seeding suggests a dissociation between misfolded protein accumulation and neurodegeneration in prion disease

Chronic neurodegenerative diseases, such as prion diseases or Alzheimer's disease, are associated with progressive accumulation of host proteins which misfold and aggregate. Neurodegeneration is restricted to specific neuronal populations which show clear accumulation of misfolded proteins, whilst neighbouring neurons remain unaffected. Such data raise interesting questions about the vulnerability of specific neuronal populations to neurodegeneration and much research has concentrated only on the mechanisms of neurodegeneration in afflicted neuronal populations. An alternative, undervalued and almost completely unstudied question however is how and why neuronal populations are resilient to neurodegeneration. One potential answer is unaffected regions do not accumulate misfolded proteins, thus mechanisms of neurodegeneration do not become activated. In this perspectives, we discuss novel data from our laboratories which demonstrate that misfolded proteins do accumulate in regions of the brain which do not show evidence of neurodegeneration and further evidence that microglial responses may define the severity of neurodegeneration.     

MAPKAP kinase 2-deficiency prevents neurons from cell death by reducing neuroinflammation--relevance in a mouse model of Parkinson's disease

The inflammatory response in the brain is closely associated with the pathogenesis of degenerative neurological disorders. A role for the p38 stress-activated protein kinase/MAPK-activated protein kinase 2 (MK2) axis in inflammation and apoptosis is well documented. Here, we provide evidence that neurodegeneration can be prevented by eliminating MK2. In primary mesencephalic neuron-glia co-cultures dopaminergic neurons from MK2-deficient (MK2−/−) mice were significantly more resistant to lipopolysaccharide-induced neurotoxicity compared with cells from wild-type mice. This neuroprotection in MK2-deficient cultures was associated with a reduced inflammatory response, especially with reduced production of the inflammatory mediators tumor necrosis factor alpha, keratinocyte-derived chemokine, interleukin-6, and nitric oxide (NO). Interestingly, in primary neuron-enriched cell cultures p38 MAPK, but not MK2, also participates in NO-mediated neuronal cell death. In the MPTP mouse model for Parkinson's disease, MK2-deficient mice show a reduced neuroinflammation and less degeneration of dopaminergic neurons in the substantia nigra after MPTP lesion compared with wild-type mice. In conclusion, our results reveal that MK2 does not directly participate in neuronal cell death, but indirectly contributes to neurodegeneration by the production of neurotoxic substances, such as NO or tumor necrosis factor alpha, from activated glia cells.     

Excitotoxic loss of post-synaptic sites is distinct temporally and mechanistically from neuronal death

Dendritic degeneration and loss of synaptic proteins are early events correlated with functional decline in neurodegenerative disease. The temporal and mechanistic relationship between synapse loss and cell death, however, remains unclear. We used confocal microscopy and image processing to count post-synaptic sites on rat hippocampal neurons by expressing post-synaptic density protein 95 fused to green fluorescent protein. Fluorescent puncta co-localized with neurotransmitter release sites, NMDA-induced Ca²⁺ increases and NMDA receptor immunoreactivity. During excitotoxic neurodegeneration, synaptic sites were lost and synaptic transmission impaired. These changes were mediated by NMDA receptors and required Ca²⁺-dependent activation of the proteasome pathway. Tracking synapses from the same cell following brief neurotoxic insult revealed transient loss followed by recovery. The time-course, concentration-dependence and mechanism for loss of post-synaptic sites were distinct from those leading to cell death. Cells expressing p14ARF, which inhibits ubiquitination of post-synaptic density protein 95 and prevents loss of synaptic sites, displayed an increased sensitivity to glutamate-induced cell death. Thus, excitotoxic synapse loss may be a disease-modifying process rather than an obligatory step leading to cell death. These results demonstrate the importance of assessing synaptic function independent of neuronal survival during neurodegeneration and indicate that this approach will be useful for identifying toxins that degrade synaptic connections and for screening for agents that protect synaptic function.     

Hassles with taking out the garbage: aggravating aggresomes

Diverse human diseases ranging from amyloidosis to neurodegenerative diseases are now recognized as 'conformational diseases' caused by protein misfolding and protein aggregation. Misfolded and aggregated proteins are usually handled in the cell through chaperone-mediated refolding, or when that is impossible, destroyed by proteasomal degradation. Recent evidence suggests that cells might have evolved a third pathway that involves the sequestration of aggregated proteins into specialized 'holding stations' called aggresomes. The aggresomal pathway provides a mechanism by which aggregated proteins form particulate (∼ 200 nm) mini-aggregates that are transported on microtubules (MTs) towards the MT organizing center (MTOC) by a process mediated by the minus-end motor protein dynein. Once at the MTOC, the individual particles pack into a single, usually spherical aggresome (1–3 μm) that surrounds the MTOC. Aggresomes are dynamic: they recruit various chaperones and proteasomes, presumably to aid in the disposal of the aggregated proteins. In addition, the formation of an aggresome is likely to activate the autophagic clearance mechanism that terminates in lysosomal degradation. Hence, the aggresome pathway may provide a novel system to deliver aggregated proteins from the cytoplasm to lysosomes for degradation. Although it is clear that many pathological states correlate with the formation of aggresomes, their causal relationships remain hotly debated. Here, we describe the current state of our knowledge of the aggresome pathway and outline the open questions that provide the focus of current research.